Metal halide perovskite toxicity effects on Arabidopsis thaliana plants are caused by iodide ions

Summary Highly efficient solar cells containing lead halide perovskites are expected to revolutionize sustainable energy production in the coming years. Perovskites are generally assumed to be toxic because of the lead (Pb), but experimental evidence to support this prediction is scarce. We tested the toxicity of the perovskite MAPbI3 (MA = CH3NH3) and several precursors in Arabidopsis thaliana plants. Both MAPbI3 and the precursor MAI hamper plant growth at concentrations above 5 μM. Lead-based precursors without iodide are only toxic above 500 μM. Iodine accumulation in Arabidopsis correlates with growth inhibition at much lower concentrations than lead. This reveals that perovskite toxicity at low concentrations is caused by iodide ions specifically, instead of lead. We calculate that toxicity thresholds for iodide, but not lead, are likely to be reached in soils upon perovskite leakage. This work stresses the importance to further understand and predict harmful effects of iodide-containing perovskites in the environment.


INTRODUCTION
Sunlight provides enough energy to fulfill the global demand, making solar cells the most promising route toward a sustainable economy. Combining solar panels with agriculture, named agri(photo)voltaics, maximizes land use, while making optimum use of the sunlight for both crops and power generation (Figure 1A). (Adeh et al., 2019) Solar cells based on both silicon and lead halide perovskites (LHPs) are considered the next-generation commercial solar cells, with potential efficiencies up to >45% (Futscher and Ehrler, 2016;Leijtens et al., 2018). However, placing such next-generation solar cells on e.g., agricultural lands raises questions about the safety of LHPs for the environment. That is, if unintentional leakage releases Pb 2+ ions in the soil, this may be harmful either to plants themselves or to humans and livestock through consumption of contaminated crops (Pourrut et al., 2011). A few studies have specifically tested the toxicity of the LHP MAPbI 3 (MA = CH 3 NH 3 ) and its supposedly less toxic tin-based counterpart on both plants and animals (Hailegnaw et al., 2015;Babayigit et al., 2016aBabayigit et al., , 2016bLi et al., 2020). These studies focused on the heavy metals, but the presence of halides also raises environmental concerns (Medrano-Macías et al., 2016;Incrocci et al., 2019), which has not been studied in plants to date.
In this work, we find that the iodide in MAPbI 3 causes greater harm to plants than the lead. Model species Arabidopsis thaliana was used to study the effect of MAPbI 3 and different perovskite precursors on the growth and development of plants. Unlike previous work on perovskite toxicity (Babayigit et al., 2016a;Li et al., 2020), we avoid acidification effects by buffering the growth media to maintain a fixed pH, so we could selectively study the effect of the perovskite and its precursors. We used controlled growth settings with a high-resolution range of salt concentrations to define the exact toxicity thresholds. We find that the presence of MAPbI 3 in the growth medium starts to affect plant performance at 5 mM and becomes significant at 10 mM. This concentration (10 mM) exceeds, according to our calculations, for a practical situation where one perovskite solar cell leaks into a similar soil area of 25 cm deep. In contrast to previous works, we conclude from experiments comparing the precursors MAI and PbI 2 to MABr and PbBr 2 , that the iodide is responsible for inhibiting Arabidopsis development, before toxicity effects of lead appear. These results show that a more rigorous assessment on the potential harmfulness of LHPs is needed and stress the importance of developing strategies to avoid halides from being released into the environment.

RESULTS AND DISCUSSION
Perovskites are fabricated from a lead halide salt, such as PbI 2 , and an organic halide salt, such as MAI ( Figure 1A):

MAI + PbI 2 / MAPbI 3
Vice versa, decomposition of MAPbI 3 leads to the formation of its precursors PbI 2 and MAI. To assess the toxicity of LHPs, we germinated seeds of Arabidopsis thaliana (ecotype Columbia-0) on media containing different concentrations of MAPbI 3 , and several precursor salts. The media were buffered at a pH of 5.8, to avoid acidification effects (Babayigit et al., 2016a). Although seed germination is not affected, both MAPbI 3 and PbI 2 significantly inhibit plant growth at the seedling stage (depicted as rosette diameter) at concentrations >10 mM ( Figures 1B and 1C). Growth inhibition stagnated from 50 mM, suggesting that no additional toxicity occurs beyond this concentration. To specify the level of lead perovskite toxicity, we grew plants at a range of MAPbI 3 concentrations around 10 mM. As shown in Figure 1D, growth is significantly inhibited by concentrations >5 mM. In addition, plants appeared bleached when treated with higher concentrations of MAPbI 3 or PbI 2 ( Figure 1B), which is supported by reduced chlorophyll levels in plants treated with over 10 mM of MAPbI 3 ( Figure 1E).
The Pb 2+ oxidation state is toxic to plants (Pourrut et al., 2011;Babayigit et al., 2016b;Gupta et al., 2020). However, at concentrations for which we observed toxicity for both MAPbI 3 and PbI 2 ( Figure 1C), lead nitrate (Pb(NO 3 ) 2 ) and another lead halide precursor (PbBr 2 ) did not affect plant growth (Figures 2A and 2B). We found these lead-containing salts to significantly impede Arabidopsis growth at concentrations >750 mM ( Figure 2C). This effect could be directly attributed to the lead, as MABr did not affect plant growth at these concentrations. Interestingly, even though growth was inhibited at high concentrations, Pb(NO 3 ) 2 and PbBr 2 did not cause plant bleaching similar to MAPbI 3 and PbI 2 (compare Figures 1B and 2C). The observation that MAPbI 3 and PbI 2 hamper Arabidopsis growth at one order of magnitude lower concentrations ( Figure 1) suggests that the iodide is at least in part responsible for this hampered growth. We repeated the growth experiments using the iodide precursor MAI and compared this to its bromide equivalent MABr ( Figure 2D). As before, we found a significant inhibition of rosette size at 50 mM of MAI. In contrast, Arabidopsis growth is not affected by any concentration of MABr up to 1000 mM ( Figures 2C and 2D).
Toxicity of MAPbI 3 starts at 10 mM, and around 50 mM for MAI ( Figure 2D), which we attribute to the threefold higher concentration of available iodide ions in the MAPbI 3 treatment. The hypothesis that the iodide is responsible for the toxicity at low MAPbI 3 concentrations is further confirmed by the absence of toxicity of MABr and PbBr 2 in the same concentration ranges ( Figure 2C).
Even though iodide is considered a (micro-) nutrient (Kiferle et al., 2021), the toxicity effects of iodide at higher concentrations are poorly understood. To confirm that iodide ions build up in the plants to reach a toxic level, we measured iodine content in Arabidopsis plants grown on different concentrations of MAPbI 3 ( Figure 3A). Iodine levels increased significantly in plants with increasing MAPbI 3 concentrations. When comparing this to rosette size, we can conclude that in-plant levels >15 ng iodine/mg fresh weight caused growth inhibition in Arabidopsis. Similar to what was shown before, low concentrations of iodide correlated with slightly induced Arabidopsis growth (although not significant, see 1 mM in Figures 1C and  1D), most probably because of its nutritional value (Kiferle et al., 2021). This trend for iodide-mediated mild growth stimulation at low concentrations and toxicity at higher concentrations is not an iScience Article Arabidopsis-specific phenotype, as it was seen before in strawberry, tomato, and various vegetable crops (Hong et al., 2009;Landini et al., 2011;Li et al., 2017).
Next, we measured Pb levels in Arabidopsis plants grown on 50 mM MAPbI 3 , or high (>500 mM) levels of MABr and PbBr 2 , see Figure 3B. As expected, Pb levels in control and MABr treated plants were extremely low or were not detectable. Plants treated with 50 mM MAPbI 3 showed some accumulation, although not significant. The rosette size data shows that growth is strongly impaired in these plants when compared to the control treatment. However, similar Pb levels (approx. 10 ng Pb/mg fresh weight) were detected in plants treated with 500 or 750 mM PbBr 2 , but do not inhibit rosette size as severely in those cases. Only after applying 1000 mM PbBr 2 , plant growth is strongly repressed. These data support that increasing iodine levels in Arabidopsis plants treated with the LHP MAPbI 3 are the main cause of toxicity and growth inhibition over increasing Pb levels. Our conclusion that the low toxicity threshold of MAPbI 3 is caused by the presence of iodide, rather than lead, stresses the importance of further investigating the environmental effects of using iodide salts in solar panels.
Likely, the extent of toxicity of these halide salts in the environment will depend on the plant species, soil type, and depth to which the salts penetrate the soil in case of leakage. We estimate that a solar panel with a 400 nm thick perovskite layer contains 0.26 mmol of Pb 2+ and 0.79 mmol of iodide (I À ) per cm 2 . If the full panel would leak and be homogeneously distributed over a water column of the same area and 10 cm deep, this would yield concentrations of 26 mM (<5 mgkg À1 soil) Pb 2+ and 79 mM iodide. Hence, the expected maximum concentration of lead in case of leakage is much lower than the concentration (>750 mM) at which it is toxic to the plant, and far below quality standards that range from 100 to 530 mgkg À1 depending on the country (Li et al., 2020). The concentration of iodide (79 mM), on the other hand, exceeds To conclude, although the perovskite community so far has mainly discussed the potential toxicity of lead (Hailegnaw et al., 2015;Babayigit et al., 2016b;Slavney et al., 2016;Li et al., 2020), we found that iodide is toxic to Arabidopsis plants at one order of magnitude lower concentrations. We further find that lead is only significantly toxic at levels far above those expected for a solar module failure. Our observations stress the importance of getting a more complete picture on the potential harmfulness of solar panels that contain LHPs, especially if these are placed on agricultural lands, and to develop strategies to prevent the release of halides into the environment. The latter should primarily be realized by careful encapsulation of the solar cells to prevent leakage, or in case of unforeseen calamities, by considering halide-tolerant plants that accumulate leaked halides in a phytoremediation approach to clean the soil (Incrocci et al., 2019).

Limitations of the study
In this study we have grown Arabidopsis seedlings in vitro, to be able to precisely apply treatments with perovskite salts. This helped to find a clear threshold for toxicity, especially iodide. In addition, we used a buffered solution to exclude effects from perovskite-induced changes in pH. However, in natural environments, where perovskites might leak from solar panels into the soil, toxicity of these compounds not only depends on their concentrations, but also on the to soil type, presence of microbiome, and other environmental factors such as water availability, pH, and the plant species. Additional experiments in natural settings would be required to test perovskite toxicity levels in natural conditions and for different plant species.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Materials availability
This study did not generate new unique reagents.
Data and code availability d Data: The data reported in this paper will be shared by the lead contact upon request.
d Code: This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Plant material and growth conditions
Arabidopsis thaliana ecotype Columbia-0 was used for all experiments. Seeds were surface-sterilized (20% bleach, 0.5%SDS), rinsed with sterile water and sown on half-strength Murashige & Skoog medium including vitamins (Duchefa Biochemie), containing 0,1% MES monohydrate buffer (Duchefa Biochemie) and 1% v/w Daishin agar (Duchefa Biochemie). Salts were added from a 1 mM stock in the desired concentration before autoclaving. The pH of all media was set at 5.8 using 0.1 N KOH to prevent any harmful acidification effects as described by Babayigit et al. (Babayigit et al., 2016a) Seeds were sown on petri dishes (9 cm diameter) containing 20 mL medium, stratified (4 C, dark) for 4 days to synchronize germination, and afterwards placed in a climate chamber (16 hours light period, 22 C, photosynthetic active radiation 150 mmol photons/m 2/ s).